1180 ORIGINAL ARTICLE J Clin Pathol: first published as 10.1136/jcp.2004.024703 on 27 October 2005. Downloaded from PCR based identification and discrimination of agents of mucormycosis and aspergillosis in paraffin wax embedded tissue R Bialek, F Konrad, J Kern, C Aepinus, L Cecenas, G M Gonzalez, G Just-Nu¨bling, B Willinger, E Presterl, C Lass-Flo¨rl, V Rickerts ............................................................................................................................... J Clin Pathol 2005;58:1180–1184. doi: 10.1136/jcp.2004.024703 Background: Invasive fungal infections are often diagnosed by histopathology without identification of the causative fungi, which show significantly different antifungal susceptibilities. Aims: To establish and evaluate a system of two seminested polymerase chain reaction (PCR) assays to identify and discriminate between agents of aspergillosis and mucormycosis in paraffin wax embedded tissue samples. Methods: DNA of 52 blinded samples from five different centres was extracted and used as a template in See end of article for two PCR assays targeting the mitochondrial aspergillosis DNA and the 18S ribosomal DNA of authors’ affiliations zygomycetes. ....................... Results: Specific fungal DNA was identified in 27 of 44 samples in accordance with a histopathological Correspondence to: diagnosis of zygomycosis or aspergillosis, respectively. Aspergillus fumigatus DNA was amplified from Dr R Bialek, Institut fu¨r one specimen of zygomycosis (diagnosed by histopathology). In four of 16 PCR negative samples no Tropenmedizin, human DNA was amplified, possibly as a result of the destruction of DNA before paraffin wax Universita¨tsklinikum embedding. In addition, eight samples from clinically suspected fungal infections (without histopatholo- Tu¨bingen, Keplerstrasse 15, 72074 Tu¨bingen, gical proof) were examined. The two PCR assays detected a concomitant infection with Absidia Germany; ralf.bialek@ corymbifera and A fumigatus in one, and infections with Rhizopus arrhizus and A fumigatus in another med.uni-tuebingen.de two cases. Accepted for publication Conclusions: The two seminested PCR assays described here can support a histopathological diagnosis of 24 March 2005 mucormycosis or aspergillosis, and can identify the infective agent, thereby optimising antifungal ....................... treatment. ilamentous fungi of the genus aspergillus and the order We developed a seminested PCR assay targeting the 18S http://jcp.bmj.com/ Mucorales cause life threatening diseases in immuno- rDNA of zygomycetes, which does not amplify genes of compromised patients and require early and effective aspergillus species, and we modified a PCR assay targeting F 12 antifungal treatment. Different mycotic infections cannot the mitochondrial DNA of Aspergillus spp, which does not be distinguished by clinical and radiological signs, but the amplify DNA from zygomycetes.20–22 The aim of our study was causative fungi show significantly different antifungal to evaluate the identification and discriminatory efficacy of susceptibilities in vitro and in vivo.1 3–6 Identification of the the two PCR assays in paraffin wax embedded tissue sections infecting fungus can be achieved by culture, but yield is low.7–10 originating from different laboratories. Invasive fungal infections are diagnosed most often by on September 30, 2021 by guest. Protected copyright. histopathology without genus or species identification. MATERIALS AND METHODS Forty two paraffin wax blocks and 10 paraffin wax embedded ‘‘Different mycotic infections cannot be distinguished by tissue sections in sterile tubes from 52 patients at risk for clinical and radiological signs, but the causative fungi invasive fungal infections were sent blinded to our laboratory show significantly different antifungal susceptibilities in in Tu¨bingen, Germany. In total, 28 biopsies of paranasal vitro and in vivo’’ sinuses, orbita, nasal mucosa, palate, or facial bones (summarised as sinus biopsies), 15 lung biopsies, five Polymerase chain reaction (PCR) assays have been biopsies of the central nervous system, three liver biopsies, introduced successfully for the identification of fungal and one skin biopsy were screened for fungal DNA. species.11 12 Fungal cultures are identified by targeting multi- Xylene (1 ml) was added to an Eppendorf tube containing copy genes such as ribosomal DNA (rDNA). However, two 5 mm sections of paraffin wax embedded tissue. The amplification and identification of fungal rDNA directly from paraffin wax was removed by incubation and washing with clinical specimens is impaired by similarities between RNA xylene and ethanol.18 DNA was extracted using the QIAamp sequences from pathogenic and contaminating fungi, ani- tissue kit as recommended by the manufacturer (Qiagen, mals, and humans.13 The increasing size of databases Hilden, Germany), modified by adding three cycles of containing fungal rDNA sequences reveals that some freezing in liquid nitrogen for one minute and boiling for formerly published primers and hybridisation probes can no five minutes to disrupt the fungal cells.16 longer be regarded as species or genus specific.14 15 To circumvent these problems, genes encoding specific proteins can be targeted, but specific proteins are unknown for most Abbreviations: PCR, polymerase chain reaction; rDNA, ribosomal of the numerous species causing mucormycosis.16–19 DNA www.jclinpath.com Zygomycetes PCR 1181 J Clin Pathol: first published as 10.1136/jcp.2004.024703 on 27 October 2005. Downloaded from 1 10 20 30 40 50 60 70 Absidia corymbifera Rhizomucor pusillus Cokeromyces recurvatus Apophysomyces elegans Saksenaea vasiformis Mucor hiemalis Rhizopus oryzae Cunninghamella bertholletiae Syncephalastrum racemosum 71 80 90 100 110 120 130 140 Absidia corymbifera Rhizomucor pusillus Cokeromyces recurvatus Apophysomyces elegans Saksenaea vasiformis Mucor hiemalis Rhizopus oryzae Cunninghamella bertholletiae Syncephalastrum racemosum 141 150 160 170 180 Absidia corymbifera Rhizomucor pusillus Cokeromyces recurvatus Apophysomyces elegans Saksenaea vasiformis Mucor hiemalis Rhizopus oryzae Cunninghamella bertholletiae Syncephalastrum racemosum Figure 1 Alignment of 18S ribosomal RNA sequences of zygomycetes amplified by inner primer set ZM1 and ZM3 based on a program according to http://jcp.bmj.com/ Corpet (http://prodes.toulouse.inra.fr/multalin/multialin.html).24 The complimentary primer regions are indicated by black bars. Black letters indicate low or missing consensus in contrast to grey letters, which indicate high consensus at a given nucleotide position. Species and GenBank accession numbers used from top to bottom: Absidia corymbifera (AF113407), Rhizomucor pusillus (AF113434), Cokeromyces recurvatus (AF113416), Apophysomyces elegans (AF113412), Saksenaea vasiformis (AF113442), Mucor hiemalis (AF113428), Rhizopus oryzae, now named R arrhizus (AF113440), Cunninghamella bertholletiae (AF113421), and Syncephalastrum racemosum (X89437). Alignment of 18S rDNA sequences from Rhizopus arrhizus The reaction mixtures of the primary PCRs consisted of (GenBank accession number AF113440) and Aspergillus 10 ml previously extracted DNA in a total volume of 50 ml, on September 30, 2021 by guest. Protected copyright. fumigatus (GenBank AB008401) by special BLAST program with final concentrations of 10mM Tris/HCl (pH 8.3), 50mM (bl2seq) at the National Center for Biotechnology KCl, and 2.5mM MgCl2 (106 Perkin-Elmer buffer II plus Information, Washington, DC, USA, revealed significant MgCl2 solution; Roche Molecular Systems, Branchburg, New differences within the V4 and V5 variable regions.11 Primers Jersey, USA); 1mM of each primer of the outer primer sets within this region show homologies to 18S rDNA of agents of (ZM1/ZM2 and P2/Asp2, respectively; Roth, Karlsruhe, zygomycosis deposited in GenBank.23 The outer primers ZM1 Germany); 1.5 U of AmpliTaq DNA polymerase (Roche); (59-ATT ACC ATG AGC AAA TCA GA-39) and ZM2 (59-TCC and 100mM of each dNTP (Promega, Madison, Wisconsin, GTC AAT TCC TTT AAG TTT C-39) are complementary to USA). Identical reaction mixtures were used for the nested nucleotide positions 711 to 730 and 1117 to 1096 (R arrhizus, PCRs except that 1 ml of the first reaction, 50mM of each AF113440), respectively. Products of the seminested reaction dNTP, and 1mM of each inner primer (ZM1/ZM3 and P1/P2) using primers ZM1 and ZM3 (59-CAA TCC AAG AAT TTC were used. All reaction mixtures were thermally cycled once ACC TCT AG-39) are 175 to 177 bp long, depending on the at 94˚C for five minutes, 35 times at 94˚C for 30 seconds, 50˚C species (fig 1).24 for 30 seconds, and 72˚C for one minute, and then once at To enhance the sensitivity of a published A fumigatus PCR, 72˚C for five minutes. a third primer was added to run a seminested PCR assay.20–22 To validate the presence of amplifiable DNA and absence of P2 (59-CTT TGG TTG CGG GTT TAG GGA TT-39) and Asp2 inhibitory substances a PCR was performed using the primer (59-GGG AGT TCA AAT CTC CCT TGG G-39) are complimen- set G1 (59-GAA GAG CCA AGG ACA GGT AC-39) and G2 (59- tary to nucleotide positions 938–916 and 738–759, respec- CAA CTT CAT CCA CGT TCA CC-39) targeting the human tively, of A fumigatus mitochondrial transfer RNA (GenBank b globin gene (nucleotides 70400–70667; accession number, accession number L37095). The product of the seminested NG_000007.3). The conditions were as described above reaction using P1 (59-GAA AGG TCA GGT GTT CGA GTC AC- except that 5 ml DNA extract and